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GCA-NAb monoclonal antibody, hybridoma cell strain and application

A hybridoma cell line and monoclonal antibody technology, applied in the fields of medicine and biochemistry, can solve problems such as mandibular osteonecrosis, atypical fractures, and poor patient compliance, and achieve the effects of increasing mRNA levels, improving bone mass, and delaying development

Active Publication Date: 2021-10-22
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defects of high price, poor patient compliance, large side effects of oral preparations on digestive tract mucosa, intravenous preparations that can cause mandibular osteonecrosis and atypical fractures in the prior art, And provide a kind of GCA-NAb monoclonal antibody, hybridoma cell line, application

Method used

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  • GCA-NAb monoclonal antibody, hybridoma cell strain and application
  • GCA-NAb monoclonal antibody, hybridoma cell strain and application
  • GCA-NAb monoclonal antibody, hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 immunogen preparation and animal immunization

[0043] (1) Amplify the sequence of the target gene as shown in SEQ ID No.2 by PCR, and insert the target gene into the corresponding expression vector; obtain a plasmid containing the correct sequence by sequencing; amplify the production of the plasmid and deliver downstream expression.

[0044] (2) Extract bacmid DNA from E coli cells containing recombinant bacmid, and obtain P1 recombinant baculovirus after infecting insect cells. The P1 generation virus was then transferred into sf9 insect cells to continue to amplify to obtain a high-titer P2 generation recombinant virus.

[0045] (3) Infect the high-five insect cells in the logarithmic growth phase with the high-titer P2 generation recombinant virus at a certain multiplicity of infection (MOI). Infected cells were further cultured at 27°C for 2-3 days to express the protein of interest.

[0046] (4) Use equilibration buffer to equilibrate the chromatog...

Embodiment 2

[0056] Example 2 blood collection and titer detection

[0057] One week after the last immunization, 50-60 μL of blood was collected through the orbital venous plexus of the mouse, and after standing at 4°C overnight, the upper serum was separated by centrifugation for inspection;

[0058] Dilute an appropriate amount of protein for detection to 5 μg / mL with coating buffer, then add 100 μL to each well of a 96-well plate with a single-channel pipette, tap the plate to mix the sample, seal it tightly with plastic wrap, and store at 4°C Coat overnight; wash the plate once with 200 μL / well of washing solution, and dry the plate; then seal the plate with 300 μL / well of blocking solution, and block for 1 hour at room temperature; wash the plate with 400 μL / well of washing solution Add the sample after 2 times (add 100 μL / well of the gradiently diluted sample and sample diluent), and add the detection antibody at the same time, add 100 μL / well into the 96-well plate, and let it reac...

Embodiment 3

[0059] Embodiment 3 fusion and screening

[0060] All the splenocytes of the immunized mice were collected, mixed with mouse myeloma cells at a ratio of 1:1, and fused by electrofusion to obtain hybridoma cells. Antigen protein was used for coating, cell supernatant was measured by ELISA method, and positive wells were selected for cloning by limiting dilution until a hybridoma cell line stably secreting monoclonal antibody was obtained. After screening, a total of 4 hybridoma cell lines were obtained. The supernatants were combined with the immunogen, that is, the monoclonal antibody GCA-Nab was successfully prepared (see figure 1 ), followed by selection of hybridoma cells with clone number MM04H for antibody preparation (preservation number is CCTCC NO: C2021182).

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Abstract

The invention discloses a GCA-NAb monoclonal antibody, a hybridoma cell strain and an application. The preservation number of the hybridoma cell strain is CCTCC NO: C2021182, the monoclonal antibody GCA-NAb secreted by the hybridoma cell strain can improve bone aging by antagonizing the influence of GCA on bone transition and fat accumulation so as to improve osteoporosis, a research basis and a theoretical basis are provided for developing medicines for treating OP, and the hybridoma cell strain can be applied to preparation of medicines for preventing and / or treating osteoporosis.

Description

technical field [0001] The invention belongs to the technical field of medicine and biochemistry, in particular, relates to a Grancalcin (GCA) neutralizing antibody (GCA neutralizing antibody, GCA-Nab) and a hybridoma cell line producing the antibody, and the antibody in Application in preparation of medicine for treating osteoporosis. Background technique [0002] Osteoporosis (Osteoporosis, OP) is a metabolic bone disease characterized by decreased bone mass, bone microarchitecture damage, increased bone fragility, and increased fracture risk. The first national osteoporosis epidemiological survey data released in 2018 showed that the prevalence of osteoporosis among people over 65 years old was as high as 32.0%, which significantly increased the risk of disability and all-cause death for the elderly, bringing Heavy economic and medical burden. [0003] At present, bone resorption inhibitors (such as bisphosphonates) are still the main clinical drugs for the treatment of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18C07K14/47C12N15/12G01N33/577G01N33/68A61K39/395A61P19/10
CPCC07K16/18C07K14/4728G01N33/577G01N33/6893A61K39/39533A61P19/10G01N2333/4727G01N2800/108
Inventor 罗湘杭李长俊
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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